There are two types of cyclic AMP (cAMP)-dependent protein kinase (PKA), type I (PKA-I) and type II (PKA-II), which contain an identical catalytic (C) subunit but have distinct regulatory (R) subunits, RI and RII, respectively. Evidence suggests that increased expression of PKA-I and its regulatory subunit (RIa) underlies tumorigenesis and tumor growth.Our studies include: (1) Antisense strategy. Antisense oligonucleotide targeted against the RIa subunit of PKA is used to demonstrate that the sequence-specific inhibition of RIa gene expression inhibits tumor growth. The results are confirmed by the use of siRNA, and the clinical utility of antisense RIa is confirmed by the findings that antisense RIa acts synergistically with cytotoxic drugs currently in use and CpG immune-modulatory oligonucleotides. Our laboratory was the first to show the genome-wide effects of antisense inhibition of gene expression in microarrays. Using in vivo tumor models of PC3M human prostate carcinoma grown in nude mice, the specificity of RIa antisense effects on gene expression signatures was critically assessed using three oligonucleotides that differed in sequence or chemical modification. Antisense treatment was found to downregulate one cluster, or signature, of genes involved in cell proliferation and upregulate another involved in cell differentiation. Importantly, these expression signatures were quiescent and unaltered in the livers of antisense-treated animals. Thus, cDNA microarray analysis defined the molecular portrait of a reverted tumor phenotype in antisense-treated regressing tumors. (2) Retroviral vector-mediated overexpression of wild-type and point-mutated R and C subunits of PKA is used to study cell growth, differentiation, and reverse transformation of tumor cells. The results obtained from the study of PC3M cells via classical biochemical analysis, DNA microarrays, and confocal microscopy demonstrated that cancer cells exhibit abnormal expression of PKA isozymes that constitute malignant phenotype, and correction of this abnormal PKA profile achieved by differential expression of the PKA R subunit genes provides a novel tumor target-based gene therapy of cancer. In OVAR-8 ovarian carcinoma cells, the molecular portraits of the distinct phenotype induced by differential R subunit gene overexpression identified the distinct disease-dependent expression profiles of clinical tumors. Many of these genes had been previously identified in clinical studies as upregulated in progressively transformed ovarian tissues as compared to normal counterparts. Analysis of pathway involvement showed active involvement of the differential signaling genes contained within the ERK/MAPK signaling pathway versus genes in the Wnt/a signaling pathway. This result suggests that perturbation of the PKA-I to PKA-II ratio in the cell is an abnormal/dangerous signal that may underlie tumorigenesis and tumor progression. This study thus suggests that protein kinase A isozyme switching in the cell might provide a novel tumor target-based gene therapy of human cancer. (3) Effect of CRE-transcription factory decoy oligonucleotides: Our previous studies showed that the CRE-decoy inhibits the CRE- and Ap1-directed transcription and inhibits cancer cell growth selectively without harming normal cell growth (Park, et al., J Biol Chem, 1999). It was found that in cancer cells, CREB (CRE-binding protein) phosphorylation by ERK/MAPK signaling overrides that by cAMP/PKA signaling, whereas, growth factor-mediated CREB phosphorylation is minimal in normal cells.